Metabolic profile of complete spinal cord injury in pons and cerebellum: A 3T 1H MRS study

The aim of this exploratory study was the assessment of the metabolic profiles of persons with complete spinal cord injury (SCI) in three region-of-interests (pons, cerebellar vermis, and cerebellar hemisphere), with magnetic resonance spectroscopy, and their correlations to clinical scores. Group differences and association between metabolic and clinical scores were examined. Fifteen people with chronic SCI (cSCI), five people with subacute SCI (sSCI) and fourteen healthy controls were included. Group comparison between cSCI and HC showed lower total N-acetyl-aspartate (tNAA) in the pons (p = 0.04) and higher glutathione (GSH) in the cerebellar vermis (p = 0.02). Choline levels in the cerebellar hemisphere were different between cSCI and HC (p = 0.02) and sSCI and HC (p = 0.02). A correlation was reported for choline containing compounds (tCho) to clinical scores in the pons (rho = − 0.55, p = 0.01). tNAA to total creatine (tNAA/tCr ratio) correlated to clinical scores in the cerebellar vermis (rho = 0.61, p = 0.004) and GSH correlated to the independence score in the cerebellar hemisphere (rho = 0.56, p = 0.01). The correlation of tNAA, tCr, tCho and GSH to clinical scores might be indicators on how well the CNS copes with the post-traumatic remodeling and might be further examined as outcome markers.

www.nature.com/scientificreports/ metabolic profiles in SCI in humans by means of MR Spectroscopy, which is a non-invasive method to determine alterations within the brain regarding neuronal metabolism. In a recently published study, we showed that the levels of glutathione (GSH) in the pons (acquired with standard point-resolved spatially localized spectroscopy (PRESS) sequence) correlated to the motor score improvements during rehabilitation in people with incomplete SCI (i.e. American spinal injury association Impairment Scale grade B, C, and D; AIS B, AIS C and AIS D) 11 .
However, this exploratory study evaluated the metabolic profile within the pons, the cerebellar vermis, and cerebellar hemispheres in people with complete (AIS A) chronic (injury > 2 years) and subacute (injury < 28 weeks) SCI. Furthermore, the advanced metabolite cycling (MC) technique was applied to acquire spectra.
Our working hypothesis presumed that total N-Acetyl-Aspartate (tNAA), reflecting neuron integrity, will show a positive correlation with the clinical neurological scores during recovery from neuronal injury. NAA has been reported as marker for stroke recovery 12 . With GSH on the other hand, being a major antioxidant and as such a neuroprotective agent, we expected that patients with high GSH levels might perform better 11 . Finally, choline containing compounds (tCho) as membrane turnover and neuro-inflammation marker was expected to be correlated with poorer scores (which has been shown in traumatic brain injury) 13,14 , and was expected to be generally low in the late stages of SCI. With total creatine (tCr) being an indicator for neuronal tissue energetics, we were particularly interested in the tNAA/tCr ratio, reflecting tissue health vs. metabolic activity. People with complete SCI were chosen as in this patient group the largest fluctuations in metabolite concentrations both immediately after SCI as well as during rehabilitation was expected 13,15 , as compared with people with lower degrees of spinal injury. From a clinical perspective, looking to establish an MRS surrogate marker for the potential of neurological rehabilitation in the patients most gravely affected from spinal injury seemed a priority over predictors of rehabilitation outcome in patients with lesser spinal injuries.

Results
Demographics. All study participants completed the full protocol, i.e. the MRI examination and clinical assessments. Two participants with SCI (one subacute, one chronic) had to be excluded due to high Hospital Anxiety and Depression Scale (HADS) score (> 7). We included 15 participants with a chronic (> 2 years) complete (AIS A) injury (cSCI, median age: 58 years, IQR range: 45-62 years; time since injury: median = 19, range: 11-35 years, 2 women), 5 participants with a subacute (< 28 weeks after injury) complete (AIS A) spinal cord injury (sSCI, median age: 41 years, IQR range: 38-47 years; years since injury: median = 12 weeks, range: 8-16 weeks, 3 women), and 14 healthy control participants (HC, median age: 40 years, range: 31-54 years, 6 women). There was a significant difference in age but not gender between the groups (p < 0.001 and p = 0.2, resp.). The demographic data are summarized in Table 1. Figure 1 illustrates the overlay of all acquired spectroscopic voxels of interest (top row) acquired in the pons (A), the cerebellar vermis (B) and the cerebellar hemisphere (C). All spectra acquired in every subgroup are frequency aligned to the water reference signal acquired with MC technique and then averaged. The averaged spectrum is fitted and the data, the fit and the residuum is shown for healthy controls (second row, number of subjects n = 14), participants with chronic SCI (third row, n = 15), and participants with subacute SCI (last row, n = 5). The median signal-to-noise ratio (SNR) in the pons was highest (cSCI: 23; sSCI: 22, HC: 26), in-between in the cerebellar vermis (cSCI: 17; sSCI: 18; HC: 19) and lowest in the cerebellar hemisphere (cSCI: 13; sSCI: 13, HC: 14). The median of the full-width at half maximum  Metabolic changes in the pons, the cerebellar vermis and the cerebellar hemisphere. We found group differences for the following metabolites (Fig. 2, Supplementary Table S1). In the pons, we report group differences in tNAA (p = 0.04, Bayes Factor BF 10 = 1.42). Post-hoc analysis showed significant differences in the concentration of tNAA between participants with chronic SCI and HC (p = 0.04, BF 10 = 2.15). In the cerebellar vermis, we report group differences for GSH (p = 0.02, BF 10 = 1.12): Post-hoc tests showed differences between participants with chronic SCI and HC (p = 0.04, BF 10 = 1. 19). Finally, in the cerebellar hemisphere, we report different tCho concentration between the groups (p = 0.01, BF 10 = 12.76). Post-hoc analysis showed a significant difference between the level of HC to participants with both chronic (p = 0.02, BF 10 = 7.24) and subacute (p = 0.02, BF 10 = 4.46) SCI.  Table S2).

Discussion
We conducted a prospective and exploratory clinical observation study in people with paraplegic complete SCI (AIS A) including two clinical groups (subacute and chronic).
SNR was sufficient and data were acquired in the pons, the cerebellar vermis and in the right side of the cerebellar hemisphere. However, the data quality was lowest in the cerebellar hemisphere, which was expected due to the complex microstructural architecture of the cerebellar hemisphere.
We reported group differences in the metabolite concentration between the three groups (cSCI, sSCI and HC) and showed correlations between metabolites in different ROIs and clinical scores.
The lower tNAA in chronic SCI is expected, as tNAA is reduced in most diseases or degenerative processes 16 , indicating less neurons per volume unit. Boesch et al. showed reduced tNAA/tCr e.g. in people with ataxia 17 . The tNAA/tCr ratio in subacute SCI might indicate ongoing transition from the tNAA/tCr levels of HC to those of cSCI.
The level of GSH is higher in chronic SCI as compared to HC and sSCI. This might be due to increased oxidative stress 18 . Higher GSH levels might indicate a better adaptation, possibly resulting in better clinical outcome.
Total choline (tCho) is significantly lower in participants with SCI indicating an effect of Wallerian degeneration beginning within the first days after injury, and, depending on the imaging method, has been reported to progress within weeks to months after imaging 19,20 . The concentration levels of the subacute and chronic SCI do not differ and the tCho concentration is lower than in HC. A possible explanation might be the reduced membrane turnover as precursor of neurodegeneration 21 .
The similar level of tCho in both SCI groups might indicate a different time scale applied for tCho compared to effects influencing the levels of tNAA and GSH.
In a recently published study, we showed that GSH correlated to motor score in incomplete SCI during rehabilitation. However, in this study, we only examined pinprick and light touch scores from the ISNCSCI assessment; since the motor score is the same in all participants, i.e. people with complete paraplegic SCI all have a motor score of 50 out of 100, indicating full motor strength in the upper body part and no motor strength in the lower extremities. PP and LT represent different aspect in perception of pain and touch 22 . We report a negative correlation between tCho and PP/LT (i.e. the lower the tCho level, the higher the clinical score). A higher level of tCho possibly indicates a higher level of inflammation, which in turn might be disadvantageous for clinical improvements. On the other hand, the higher the level of tNAA/tCr indicating more viable neurons, the better the clinical score, confirming the finding of a positive correlation between tNAA/tCr in the cerebellar vermis and PP/LT. www.nature.com/scientificreports/ Interestingly, we found a positive correlation between the level of GSH in the cerebellar hemisphere to total SCIM score and SCIM part 1 (self-care) and 3a (bed mobility and transfers), the scores ranging from 0 to 100 (total SCIM), 0-20 (part 1) and 0-10 (part 3a). The correlation of GSH in the cerebellar hemisphere to the total SCIM score might be explained by the anti-oxidative effect, thus higher GSH levels reflect a state of higher alertness against oxidative stress and therefore better clinical performance. Finally, the positive correlation of tNAA/tCr as shown in Fig. 4D and thus the negative correlation of tCr to SCIM part 2 (sphincter management) might be explained by a possible overcompensation, i.e. higher activity of neurons needed to achieve previous abilities. In summary, the higher GSH and tNAA and the lower tCr and tCho, the better the clinical outcome in people with complete paraplegic SCI. Further study might examine these biomarkers as possible therapy outcome measurement to improve rehabilitation therapy.
Our study had important limitations: The overall number of participants was small, especially in the subacute group (with only five valid data sets). However, we conducted non-parametric group comparison tests and used non-parametric correlation analysis to address this point. In addition to frequentist statistics (null hypothesis significance testing), we included Bayesian statistics to quantify the evidence from the data itself for or against the null hypothesis as has been previously described 11 , which does not require correction for multiple testing.
We acquired spectra in three distinct regions of interest with single voxel spectroscopy using the advanced MC technique. A future study might apply multi-voxel techniques to further investigate the effect of complete paraplegic SCI. www.nature.com/scientificreports/ In conclusion, we showed group differences for the metabolites tNAA, GSH and tCho in data acquired in complete, paraplegic SCI and HC with means of short-echo MR spectroscopy. In addition, tNAA, tCr and tCho correlated with the pinprick and light touch scores in the pons and cerebellar vermis. Furthermore, GSH in the cerebellar hemisphere correlated with the SCIM score, which measures patients' independence. Further studies might include complete tetraplegic patients to examine the influence of the level of injury on the degree of metabolite-alteration in the cerebellar and pontine regions.

Materials and methods
Institutional Review Board Approval. The local ethics committee northwest and central Switzerland (EKNZ) approved the following study (2019-01624), which was conducted in accordance with the Declaration of Helsinki. All participants were informed about the aim and procedure of this prospective study and gave written informed consent prior to study enrollment.
We used the MC 26,27 PRESS technique (TR/TE: 2000/30, spectral bandwidth: 2000 Hz, readout duration: 512 ms). The MRS sequences included a non-water suppressed reference shim scan (i.e. the water reference signal) and a MC sequence (i.e. the metabolite signal with alternative cycled inversion pulse) including higher-order shimming, broadband outer volume suppression and inner volume saturation pulses to minimize the chemical shift displacement artefact as described previously 6,27 .
The basis set for spectral fitting included the following metabolites: N-acetyl-aspartate and N-acetyl-aspartylglutamate (NAA + NAAG = tNAA), GSH, glutamate and glutamine (Glu + Gln = Glx), glycerophosphochline and phosphocholine (GPC + PCh = tCho), Cr, scyllo-Inositol (sI) and myo-Inositol (mI). Metabolites were included based on the MRS consensus recommendations 32,33 . All MR spectroscopy acquisitions were performed by a trained spectroscopist (last author with 8 years of experience). Data analysis were performed by using an automated post-processing pipeline.
Statistical analysis. We used R software (version 4.0.2, R Core Team 2020) with the tidyverse package 34 .
Boxplots show data including medians and quartiles; medians and ranges are reported in the text. Group differences were assessed by using Kruskal-Wallis tests followed by Dunn's test for pairwise multiple comparisons. Correlation analyses were performed using Spearman rank correlation. Linear regression model included clinical scores and metabolite concentration measurements. The confidence interval was set to 95% and p < 0.05 was assumed to indicate a significant difference. Age was included in the linear regression models as a covariate. Due to the exploratory nature of the study, no adjustments for multiplicity were applied. We included Bayesian statistics to quantify the evidence of the null hypothesis and the alternative hypothesis. We calculated the Bayes factor BF 10 reflecting the support of the alternative hypothesis over the null hypotheses with the R package bayestestR (version 0.13.0) 35 .

Data availability
The data that support the findings of this study are available from the corresponding author upon reasonable request. www.nature.com/scientificreports/ Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/.